The goal of this proposed research is to understand the cause of baculovirus infected insect cell death. It is expected that this will lead to the development of methodologies to extend the life span of baculovirus infected insect cells and to an improved understanding of viral cytotoxicity, in general. The hypothesis upon which this research is based is that oxidative stress and cathepsin expression contribute appreciably to baculovirus cytotoxicity. Reactive oxygen species (ROS, e.g., superoxide radical (SR) and hydrogen peroxide (HP)) overexpression, while benefiting early stages of viral replication, can also lead to premature cell death through lipid oxidation that results in membrane degradation. For example, it has been demonstrated that the concentrations of superoxide radical, oxidized proteins, and oxidized lipids increase as the baculovirus infection proceeds. In addition, it has been shown that manganese superoxide dismutase (MnSOD, which converts SR to HP) overexpression extends the life span of baculovirus infected insect cells. Furthermore, baculoviruses express cathepsin that degrades cellular actin, thereby compromising cellular integrity. The specific aims that will be carried out to test the hypothesis of this proposed project are (1) evaluate the role of oxidative stress in baculovirus cytotoxicity and (2) evaluate the role of cathepsin expression in baculovirus cytotoxicity. The role of oxidative stress will be evaluated by infecting metabolically engineered cells that target superoxide dismutases (which convert SR to HP) and catalase and/or ascorbate peroxidase (which convert HP to water) expression to the intracellular locations of SR and HP accumulation, respectively. The role of cathepsin will be evaluated by infecting cells with a modified baculovirus lacking the cathepsin gene. In addition, the synergistic effects of oxidative stress and cathepsin expression will be investigated by infecting the metabolically engineered cells with the baculovirus lacking the cathepsin gene. The proposed research will advance the understanding of the role of oxidative stress and cathepsin expression in baculovirus cytotoxicity. The role of oxidative stress is particularly important since its role in viral cytotoxicity, in general, is not very well understood. It is possible that understanding this phenomenon could lead to the development of novel antiviral drugs.